Click chemistry facilitates direct labelling and super-resolution imaging of nucleic acids and proteins

Raulf, Anika; Spahn, Christoph; Zessin, Patrick J. M.; Finan, Kieran; Bernhardt, Stefan; Heckel, Alexander; Heilemann, Mike

doi:10.1039/c4ra01027b

Cited by 49 publications

(40 citation statements)

References 44 publications

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“…In the absence of infection, bright EdU fluorescence was detected in the nuclei of about one-third of the unsynchronized cells, and the pattern of granular perinuclear and intranuclear labeling (Fig. 1A) was typical of mid-S phase (70,71). Nuclear EdU fluorescence intensity decreased markedly after infection, although it was still detectable at a low level in some cells.…”

Section: Resultsmentioning

confidence: 99%

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

Senkevich

Katsafanas

Weisberg

et al. 2017

J Virol

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Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2=-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor-or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate-and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases II␣ and II␤ and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected. IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNAdependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases II␣ and II␤ and PCNA, which were found associated with nascent DNA.

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Section: Resultsmentioning

confidence: 99%

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

Senkevich

Katsafanas

Weisberg

et al. 2017

J Virol

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“…New enzymatic labeling schemes, several tag‐technologies including SNAP‐, HALO‐, CLIP‐ and His‐tags, or click chemistry combine direct labeling with high specificity and the possibility to use bright, photostable fluorophores, for example, organic dyes or quantum dots 50. 51, 108–111…”

Section: Discussion and Outlookmentioning

confidence: 99%

Single‐Molecule Methods to Study Membrane Receptor Oligomerization

2014

Self Cite

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Membrane receptors control fundamental cellular processes. Binding of a specific ligand to a receptor initiates communication through the membrane and activation of signaling cascades. This activation process often leads to a spatial rearrangement of receptors in the membrane at the molecular level. Single-molecule techniques contributed significantly to the understanding of receptor organization and rearrangement in membranes. Here, we review four prominent single-molecule techniques that have been applied to membrane receptors, namely, stepwise photobleaching, Förster resonance energy transfer, sub-diffraction localization microscopy and co-tracking. We discuss the requirements, benefits and limitations of each technique, discuss target labeling, present a selection of applications and results and compare the different methodologies.

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“…Super-resolution microscopy methods can be broadly divided into two categories: those that are based on patterning the illumination light, such as Saturated Structured Illumination Microscopy -(S)SIM [18,19] and Stimulated Emission Depletion (STED) [20] or those that are based on single molecule detection and localization, such as Stochastic Optical Reconstruction Microscopy -STORM [21] and (Fluorescence) Photoactivation Localization Microscopy -PALM and fPALM [22,23]. Most of these methods have been applied to image chromatin and nuclear organization at high resolution [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]. Below, we summarize the concept behind each one of these methods as well as their strengths and weaknesses and overview how each method has been used to visualize nuclear organization.…”

Section: Super-resolution Methodsmentioning

confidence: 99%

Advanced microscopy methods for visualizing chromatin structure

Lakadamyali

Cosma

2015

FEBS Letters

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a b s t r a c tIn the recent years it has become clear that our genome is not randomly organized and its architecture is tightly linked to its function. While genomic studies have given much insight into genome organization, they mostly rely on averaging over large populations of cells, are not compatible with living cells and have limited resolution. For studying genome organization in single living cells, microscopy is indispensable. In addition, the visualization of biological structures helps to understand their function. Up to now, fluorescence microscopy has allowed us to probe the larger scale organization of chromosome territories in the micron length scales, however, the smaller length scales remained invisible due to the diffraction limited spatial resolution of fluorescence microscopy. Thanks to the advent of super-resolution microscopy methods, we are finally starting to be able to probe the nanoscale organization of chromatin in vivo and these methods have the potential to greatly advance our knowledge about chromatin structure and function relationship.

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Click chemistry facilitates direct labelling and super-resolution imaging of nucleic acids and proteins

Abstract: We demonstrate super-resolution imaging of proteins and nucleic acids that were densely labelled with fluorophores using the concept of “click chemistry”.

Cited by 49 publications

References 44 publications

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

Single‐Molecule Methods to Study Membrane Receptor Oligomerization

Advanced microscopy methods for visualizing chromatin structure

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